Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors

Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5–13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

: Properties of dark-LUMABS-HIV variants S6 Figure S1: Unspecific binding of SmBiT peptides to BSA S7 Figure S2: Kinetics of SmBiT86 peptide binding S7 Figure S3: Overview of Limit of Detection (LoD) determination S8 Figure S4: Comparison of full-length NanoLuc and NanoBiT S9 Figure S5: Expanded thermodynamic equilibrium model S9 Figure S6: Simulations of K D SmBiT / K D DarkBiT changes S10 Figure S7: Comparison of incubation times S10 Figure S8: Kinetic measurements and k obs plots S11 Figure S9: Kinetic behavior of anti-HIV1-p17 sensors S12 Figure S10: Kinetic behavior of 101F sensors S13 Figure S11: Simulations of K D Ab / C eff Ab changes S13 Peptide & Protein sequences S14 References S17

Materials
Peptides with acetylated N-terminus and amidated C-terminus were purchased from GenScript (SB86: VSGWRLFKKIS, ≥75% purity; DarkBiT 101: SVTGYALFEKESG, ≥75% purity). Terminal S and G were added to the DarkBiT peptide to account for linker amino acids in fusion proteins. NanoGlo substrate was purchased from Promega. Unless stated otherwise, chemicals were purchased from Sigma.

Protein purification
For purification of dark-LUMABS variants and calibrator luciferase, E. coli BL21 (DE3) cells were transformed with corresponding pET28a(+) plasmids. Cells were grown in LB medium supplemented with 50 µg/ml kanamycin at 37 °C at 180 rpm in a shaking incubator. Large cultures (750 ml) in 2 l baffled flasks were inoculated with corresponding overnight cultures and induced with IPTG at OD 600 0.6-0.8. Proteins were expressed overnight at 18 °C. Harvested cells were lysed with BugBuster reagent (Novagen) supplemented with Benzonase (Merck), and proteins were purified with Ni-NTA chromatography followed by Strep-Tactin XT (iba) using gravity flow columns. Protein purity was confirmed by reducing SDS-PAGE and concentrations calculated using A 280 nanodrop measurements and the corresponding extinction coefficients (based on protein sequence). Proteins in Strep-Tactin XT elution buffer (150 mM NaCl, 100 mM Tris-Cl pH 8, 1 mM EDTA, 50 mM D-biotin) were aliquoted, flash frozen in liquid N 2 and stored at -70 °C. NB-LUMABS-HIV6 was expressed and prepared as described in (3), 3E2H.37-LUMABS was expressed and purified as described in (4).

Thermodynamic equilibrium scheme
The equilibrium scheme that was devised in the main text Figure 5A is adapted from previous works on the LUMABS and RAPPID platforms (5,6). The equilibrium constants are defined as follows: K 1 , intramolecular release of the DarkBiT from LgBiT; K 2 , monovalent antibody binding based on the 4 possible binding combinations and the monovalent affinity of the epitope K D Ab ; K 3 , intramolecular binding of SmBiT to LgBiT; K 4 , formation of the bivalent complex. The equilibrium constants take effective concentrations (C eff ) into account that result from intramolecular linkage (for DarkBiT and SmBiT) or intermolecular recruitment (for the bivalent complex formation). The overall apparent dissociation constant K D,app is the product of the inversed equilibrium constants K 1 to K 4 (5) and can be written as With all other parameters known, C eff Ab for each sensor could be calculated from this equation.

Calculation of effective concentrations
The term effective concentration C eff connects an intramolecular affinity to an intermolecular affinity (7) and is defined as C eff = K D inter / K D intra C eff DarkBiT was derived from competition experiments as 1.73 mM (see main text). As the SmBiT is connected to the LgBiT with a well-understood, flexible GGS linker, the C eff SmBiT was determined with the effective concentration calculator app (8), considering a persistence length of 3.7 Å (9) and distance of 30 Å between the N-terminus of LgBiT and the binding site of SmBiT measured using Chimera and the PDB file 5IBO. The app gave a C eff SmBiT of 4.66 mM.

Thermodynamic equilibrium simulations using the expanded model
To understand how the hook effect is affected by changes in the sensors intrinsic properties, simulations were performed with a general python model framework developed by Geertjens et al. (10) in the Spyder environment. The framework is freely available under DOI: 10.5281/zenodo.5531622. The mass balance equations used to build the model derive from the expanded thermodynamic equilibrium scheme shown in Figure S3, and were written in python as:

Titrations
Unless specified otherwise, all titrations were performed in 1xPBS (10 mM phosphate buffer, 2.7 mM KCl and 137 mM NaCl, pH 7.4) supplemented with 1 mg/ml BSA to avoid unspecific adsorption of proteins to surfaces. As additive, BSA worked better and gave higher signal intensities than Tween20 in all experiments except titrations with SmBiT peptides. Assay components were diluted in the same buffer before measurements. Unless stated otherwise, the sensor proteins were incubated with different concentrations of target antibody for 16 h at 4 °C to allow equilibration, before NanoGlo substrate (furimazine) was added from a freshly prepared 10x stock directly before the measurement to obtain the final volume and concentrations. Unless stated otherwise, the final NanoGlo dilution during the measurements was 1:2000. Binding assays were performed in white 364-well plates (flat bottom, Nunc, ThermoFisher) and a total volume of 20 µl with n=3 technical replicates. Unless stated otherwise, bioluminescence at 458 nm was determined from bioluminescence spectra (398-653 nm, integration time 100 ms) measured at 20 °C in a TECAN Spark 10M. Resulting binding curves were fitted to the standard binding model With Max being the signal at saturated sensor (mean of n=3) and Min the signal in absence of the target (mean of n=3). DR uncertainties were propagated from the standard deviation of Max and Min (s(Max) and s(Min), respectively) using (11)

DarkBiT101 affinity
For data evaluation of competitive binding based on Motulsky and Neubig (12), DarkBiT101 concentrations were converted to logarithmic values and the bioluminescence signals were fitted to The background was constrained to positive values. Fitting was performed in Origin 2020. The K i was then determined from with the concentration of active SmBiT86 [SmBiT86] and its K D for LgBiT binding that was determined in a parallel binding experiment (main text Figure 2A).

Fitting of single exponential binding processes
To determine the observed rate constants of the association reactions, k obs , in kinetic experiments, the traces were fit to a single exponential binding process using (13) = - Where Y is the observed blue-to-green ratio, Y eq is the final equilibrium value of the blue-to-green ratio, k obs is the observed rate constant of the respective association reaction, and t is the time in minutes. Table S1 Properties of dark-LUMABS-HIV variants tested in this study. The titration curves in Figure 5B (main text) were fitted until the start of the hook effect with        The kinetic traces were fitted to a single exponential binding process using Y = -Y eq *e^(-k obs *t)+Y eq according to (13). The k obs values of the fitting results were plotted as a function of antibody concentrations to check for concentration dependency.